Improved Boronate Affinity Electrophoresis by Optimization of the Running Buffer for a Single-step Separation of piRNA from Mouse Testis Total RNA

Sato, Yusuke; Iwasawa, Daijiro; Hui, Kuo Ping; Nakagomi, Rena; Nishizawa, Seiichi

doi:10.2116/analsci.17n024

Cited by 5 publications

(4 citation statements)

References 27 publications

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“…In addition to standard electrophoresis, we have also analyzed the samples in polyacrylamide gels containing 1% acryloyl phenyl boronic acid (APB). ABP has been used to differentiate the mobility of capped and uncapped RNAs due to interactions with cis-diol groups, which are present at the RNA 3′-ends and optionally at the 5′-ends if the RNA is 5′ capped (Nubel et al, 2017;Sato et al, 2018). Hence, slower migration through the APB gels is expected for capped RNAs (due to one more cis-diol function within the RNA) compared to uncapped RNA, allowing an easier separation and assessment of incorporation.…”

Section: Capping Efficiency Using G (φ 65) Promotermentioning

confidence: 99%

Preparation of RNAs with non-canonical 5′ ends using novel di- and trinucleotide reagents for co-transcriptional capping

Depaix

Grudzien-Nogalska

Fedorczyk

et al. 2022

Front. Mol. Biosci.

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Many eukaryotic and some bacterial RNAs are modified at the 5′ end by the addition of cap structures. In addition to the classic 7-methylguanosine 5′ cap in eukaryotic mRNA, several non-canonical caps have recently been identified, including NAD-linked, FAD-linked, and UDP-glucose-linked RNAs. However, studies of the biochemical properties of these caps are impaired by the limited access to in vitro transcribed RNA probes of high quality, as the typical capping efficiencies with NAD or FAD dinucleotides achieved in the presence of T7 polymerase rarely exceed 50%, and pyrimidine derivatives are not incorporated because of promoter sequence limitations. To address this issue, we developed a series of di- and trinucleotide capping reagents and in vitro transcription conditions to provide straightforward access to unconventionally capped RNAs with improved 5′-end homogeneity. We show that because of the transcription start site flexibility of T7 polymerase, R1ppApG-type structures (where R1 is either nicotinamide riboside or riboflavin) are efficiently incorporated into RNA during transcription from dsDNA templates containing both φ 6.5 and φ 2.5 promoters and enable high capping efficiencies (∼90%). Moreover, uridine-initiated RNAs are accessible by transcription from templates containing the φ 6.5 promoter performed in the presence of R2ppUpG-type initiating nucleotides (where R2 is a sugar or phosphate moiety). We successfully employed this strategy to obtain several nucleotide-sugar-capped and uncapped RNAs. The capping reagents developed herein provide easy access to chemical probes to elucidate the biological roles of non-canonical RNA 5′ capping.

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Section: Capping Efficiency Using G (φ 65) Promotermentioning

confidence: 99%

Preparation of RNAs with non-canonical 5′ ends using novel di- and trinucleotide reagents for co-transcriptional capping

Depaix

Grudzien-Nogalska

Fedorczyk

et al. 2022

Front. Mol. Biosci.

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“…Direct detection by oxidation reaction is very important because RNA-derived substances have been actively researched recently. [31][32] Moreover, uridic and inosic acid are also umami components. These food components are typically determined by using HPLC-based techniques.…”

Section: Detection Ability Of Biomoleculesmentioning

confidence: 99%

Controlling Surface Oxygen Concentration of a Nanocarbon Film Electrode for Improvement of Target Analytes

et al. 2019

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A nanocarbon film consisting of nanocrystallites with mixed sp 2 and sp 3 bonds formed by unbalanced magnetron sputtering was studied with respect to the changes in characteristics caused by surface oxygen concentration. Electrochemical pretreatment (ECP) was conducted to change the surface oxygen concentration of the nanocarbon film. X-ray photoelectron spectroscopy (XPS) measurements revealed that nanocarbon films with different amounts of surface oxygen could be prepared. In addition, we observed no significant increase of surface roughness (R a) at the angstrom level after ECP, owing to stable structure containing 40 % of sp 3 bonds. Electrode characteristics including potential window, and electrochemical properties for some redox species such as Ru(NH 3) 6 3+/2+ , Fe(CN) 6 3-/4and some biomolecules were investigated. The anodic potential limit became wider and ΔE p of Fe(CN) 6 3-/4became smaller at the treated nanocarbon film electrode than those of the as-deposited nanocarbon film electrode. Based on these results, we realized to measure uridylic acid (UMP) and inosine triphosphate (ITP) with high oxidation potential by direct oxidation, which was difficult to measure at the as-deposited nanocarbon film electrode.

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“…Catechol is a kind of cis-diol containing compound. Until now, many conventional boronate affinity analytical methods have been used to detect cis-diol containing compounds, including liquid chromatography, [11][12][13] chemiluminescence, [14] capillary electrophoresis, [15][16][17] etc. The main objective of the present research was to separate cis-diol containing substance from non-cis-diol containing substances.…”

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confidence: 99%

A hybrid boronate affinity probe for the selective detection of cis‐diol containing compounds in tea beverages

et al. 2022

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UiO-66-NH 2 nanocomposite was post-modified with 4-mercaptophenylboronic acid (MPBA) by the method of in situ hybridization reaction. The hybrid boronate affinity material UiO-NH 2 @P (TEPIC-co-MPBA) was characterized by scanning electron microscopy, X-ray diffraction and Fourier-transform infrared spectroscopy. The material was applied as fluorescent probe for the detection of cis-diol containing compounds based on the boronate affinity mechanism, and exhibited high specific selectively. The proposed method exhibited good linearity for the detection of catechol in the range of 0.50 to 8.00 μg ml À1 . The detection limit was 0.13 μg ml À1 .The tactic was successfully applied to analyze the total polyphenols in tea beverages for catechol, and relative recovery was in 98.86-106.00%. Therefore, this work provided a promising strategy for the recognition of cis-diol containing compounds.boronate affinity, cis-diol containing compounds, detection, UiO-NH 2 hybrid material | INTRODUCTIONGreen tea is one of the most popular drinks in the world. [1] Tea polyphenols, such as catechins and theaflavins, are important functional constituents in green tea. Catechins can effectively inhibit or delay

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Improved Boronate Affinity Electrophoresis by Optimization of the Running Buffer for a Single-step Separation of piRNA from Mouse Testis Total RNA

Cited by 5 publications

References 27 publications

Preparation of RNAs with non-canonical 5′ ends using novel di- and trinucleotide reagents for co-transcriptional capping

Preparation of RNAs with non-canonical 5′ ends using novel di- and trinucleotide reagents for co-transcriptional capping

Controlling Surface Oxygen Concentration of a Nanocarbon Film Electrode for Improvement of Target Analytes

A hybrid boronate affinity probe for the selective detection of cis‐diol containing compounds in tea beverages

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